This disclosure provides improved methods for enumeration of lactic acid bacteria, such as Lactobacilli or Bifidobacteria, within a freeze-dried powder. In conventional enumeration methods, powdered lactic acid bacteria, which optimally grows under acidic conditions, is rehydrated in a culture broth having an acidic pH (e.g., Difco® MRS) to form a rehydrated sample. The sample is held at room temperature for 30 minutes, and is then serially diluted to form a series of serial dilutions. The serial dilutions are then plated with an agar composition to form a series of plates, which are incubated under anaerobic conditions for 72 hours to form colonies. Excessive incubation time may cause excessive radial growth of the colonies, such that the colonies grow into one another making them difficult to count. After incubation, the colonies on each plate are counted, and the number of microorganisms in the powder is quantified based on a correlation between the number of colonies and the dilution for each plate.
Some freeze-dried lactic acid powders may contain additional components, such as polymeric materials (e.g., alginate) that may delay the release of the microorganisms in certain environments. It has been unexpectedly found that the use of conventional methods for enumeration of microorganisms may be inaccurate when the powder contains these polymeric materials. Without wishing to be bound by theory, it is possible that the polymeric materials may lead to the agglomeration of colony forming units (CFUs) when the powder is reconstituted, diluted and plated, thereby leading to an undercounting of viable microorganisms. For example, if a sample having CFUs contains a polymeric material in sufficient quantity to cause CFUs to be bound to other CFUs at the time of plating, then conventional methods may undercount the actual number of CFUs. Accordingly, a need exists for improved enumeration methods that overcome the aforementioned shortcomings.